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Author Topic: New to ABC staining  (Read 2591 times)

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Offline sophiamariapickle

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New to ABC staining
« on: January 19, 2005, 12:23:08 AM »
Hi,

I have a few questions: I just starting ABC staining on rat brain tissue (substantia nigra and striatum for tyrosine hydroxylase). I have the Vector Elite kit and their horse serum and biotinylated secondary Ab and their DAB stuff. So far, I have followed the given protocol exactly but  I can't see any of my staining and there is TONS of background. I've tried increasing the washing time and primary AB incubation time.

I've read something about H202 blocking. would i have to do this for free floating sections? and what about avidin/biotin blocking. can someone explain what these two do? and how can i fix my staining problems!!!???  :shock:

New to ABC staining
« on: January 19, 2005, 12:23:08 AM »

Offline immunoem

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    • http://www.bath.ac.uk/~bssiwj/
New to ABC staining
« Reply #1 on: January 19, 2005, 04:24:21 AM »
Hi,
you shouldn't need to do a peroxide or avidin/biotin block. I suspect that the problem lies with the detection reagent concentrations. Vector's ABC protocol is optimised for wax sections and usually needs to be tweaked for thicker samples, such as vibratome sections.
I am guessing that you are using 4% PFA-fixed vibratome sections, in which case try the following method:
http://www.bath.ac.uk/~bssiwj/files/IHC%20-%20peroxidase%20method.pdf
We find that the sodium borohydride step is not necessary for TH labelling. You will see that the concentration of the biotinylated antibody (1:1000) is lower than suggested, as is the ABC reagent (again 1:1000 for both A and B componants).
We've used this method succesfully for several different anti-TH antibodies and it works fine. If you still get high background, try diluting out the primary antbody (we find most work around 1:1000).
Good luck.
Ian

Offline Jossy

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New to ABC staining
« Reply #2 on: January 19, 2005, 06:38:41 PM »
I worked for 4 years with rat brain tissue (septum and hippocampus) and my experience is that peroxide bloking step is essential, but the avidin-biotin bloking step don't. The sodium borohydride step (as Ian says) is necessary only if you want to make a immunofluorescence, because the free aldehydes has autofluorescence.
If your sections are 30-50 micrometers thick, 0.3-0.5% H2O2, 30 minutes, must to be enough.
Try incubating the first antibody O.N at 4C, and use TBS (50mM tris,140 mM NaCl, pH 7.6) for washing steps (3x10min). Don't use Tx-100 or another detergent after 1 Ab (only in serum blocking solution and 1 Ab solution).
Here is my general protocol for IHC....Good Luck :D

IHQ
Wash 15 min in TBS at RT
Incubate 15 min in 0.3% H2O2 (in TBS) at RT
Wash 5 min in TBS at RT
Incubate 1 hr in TBS + 0.2% Triton x-100 / 5% NS at RT
Incubate with primary antibody in TBS + 0.2% Triton x-100 / 2% NS at 4 C,  O.N.
Wash 3 times in TBS , 15 min each, at RT
Incubate with secondary biotinylated antibody in TBS / 0.1% NS, 2 hrs at RT
Wash 3 times in TBS , 15 min each, at RT
Incubate one and a half hour with ABC Kit (1:200) diluted in TBS, at RT (mix 30 min before use)
Wash 3 times in TBS , 15 min each, at RT
Incubate in 0.04% DAB + 0.01% H2O2 + NiCl2 0.06%, 10 min at RT (discard to chlorine solution)
Wash 3 times in TBS , 15 min each, at RT (discard to chlorine solution)
ossy
jossy@neurobio.pitt.edu

New to ABC staining
« Reply #2 on: January 19, 2005, 06:38:41 PM »